Balancing physicochemical properties and sensory properties is one of the key points in expanding edible packaging applications. The work consisted of two parts, one was to investigate the effects of cellulose nanocrystals (CNC) on the packaging-related properties of whey protein isolate films with natural colorants  under freeze-thaw (FT) conditions; the other was to test oral tactility and visual sensory properties of the edible films and their overall acceptability in packed ice cream. FT treatment reduced the mechanical strength and moisture content and increased the water vapor permeability of the films, as water-phase transformation not only disrupted hydrogen bonds but also the film network structure through physical stress. The oral tactility produced by CNC and the visual effect produced by colorants could affect participants’ preference for edible films. This study provides a good reference for the consumer-driven product development of packaged low-temperature products. Edible packaging materials can enter the mouth along with food and be safely consumed after oral processing through mastication, agglomeration, and lubrication via saliva . UnderstColored edible packaging gives intuitive visual effects and can efficiently reduce the excessive addition of coloring agents to food itself, such as in individually packaged ice cream or ready-to-eat jelly. At present, after appropriate molecular modification or microencapsulation, some natural colorants, such as curcumin, lycopene, and phycocyanin, are gradually being introduced to dye edible packaging instead of synthetic colorants, to improve appearance, ensure food safety, and promote consumer acceptance ,3,4. NevIn edible materials, whey protein isolate (WPI) has become among the most suitable substrate for edible composite films due to its high nutritional quality, strong film-forming ability, and potential to effectively carry different functional components ,6. ConsiCellulose nanocrystals (CNC) from plant fibers are generally recognized as safe (GRAS) food additives and widely used in food products as a reinforcing agent, dietary fiber supplement, and low-calorie food filler ,9. RecenHence, the aim of this work was to search colored edible WPI-CNC packaging films suitable for packaging food in a low temperature environment and give rise to consumer preferences by investigating the effects of CNC on the physicochemical and sensory properties of films containing curcumin, phycocyanin, and lycopene under FT conditions. In particular, the oral and visual sensory performance of colored edible films in packed ice cream were emphatically investigated. This study is crucial in promoting the practical applications of edible packaging.WPI powder with 90% protein content on a dry weight basis was purchased from Fonterra Co-operative Group . CNC aqueous dispersion  was provided by Guilin Qihong Technology Co., Ltd. . CNC dispersion before use was ultrasonicated by an ultrasonic processor  with 300 W for 10 min to ensure good homogeneity. Food-grade NaOH was purchased from Angel Yeast corporation . Food-grade HCl was purchased from Kunfeng Chemical Co., Ltd. . Curcumin (90%) was purchased from Tekang Biotechnology Co., Ltd. . Water-soluble modified lycopene  was obtained from Xihai Biotechnology Co., Ltd. . Phycocyanin (purity (A620 nm/A280 nm) >2.5) was obtained from Binmei Biotechnology Co., Ltd. . Distilled water was used for all sample preparations.Briefly, 5.0 g WPI and 2.5 g glycerol (as plasticizer) were dissolved in 50 mL of distilled water with continuous stirring for 2 h at room temperature as the stock solution. In addition, all sample preparations had the same stirring duration to prevent the influence of stirring parameters.Since the addition of curcumin requires a specific alkali pretreatment process, to ensure comparability between films, except blank sample (WPI-1), all stock film-forming solutions were subjected to the same alkali treatment process so that the final pH values and ionic strengths remained constant.The WPI-1 film-forming solution was prepared as previously reported by Chai et al. , with soPreparation of the WPI-2 film-forming solution was performed as described by Taghavi Kevij et al. . The pH w/w) of WPI dry weight) by preliminary experiments (data shown in w/w)) were added to the WPI-2 film-forming solution, and the resulting WPI-3 film-forming solution was stirred continuously for 12 h.After determining the appropriate CNC content  relative to WPI) or modified lycopene  relative to WPI) under agitation in the dark for 1 h.WPI-3-phycocyanin (WPI-3-Phy) or WPI-3-modified lycopene (WPI-3-Lyc) film-forming solution: WPI-3 film-forming solution was added with phycocyanin  relative to WPI) was added to the solution under agitation for 30 min in the dark, and the pH was gradually reduced to 7.0 with 3.0 M HCl; 5% CNC were added to this solution, and it was stirred in the dark for 12 h.WPI-3-Cur film-forming solution: This solution was prepared according to the method proposed by Pan et al. , with moAfter eliminating tiny particles and bubbles by ultrasonication, film-forming solutions were cast on Plexiglas plates (9-cm inner diameter) and dried at 40 °C for 24 h. Films were peeled off and equilibrated at 23 ± 2 °C and 50% ± 8% relative humidity  for at least 72 h .Films were subjected to FT seven cycles  and thawing for 1 h at 22 ± 3 °C in the dark). The preservation duration of non-freeze-thaw (non-FT) films and the films prepared under seven continuous freeze-thaw cycles (7-FT) was the same .MC was determined by recording mass loss after oven-drying of small film strips (2-cm diameter) at 105 °C for 24 h and calculated as follows [m is the mass loss after drying and m is the initial mass. Five replicate measurements were performed for each film.Average film thickness  was determined for each type of film using a digital micrometer . The  follows :(1)MC(%)WVP was calculated as follows:m is mass gain (g), A is the area of the exposed film (m2), and The WVP was determined by sealing cups containing calcium chloride (0% RH) with the films and placing the cups in a desiccator containing saturated NaCl solution  . The weiSR values were calculated as follows [Small film strips were pre-dried in an oven at 105 °C for 2 h to determine the initial dry weight ( follows :(3)SR(%)TS) and elongation at break (EAB) of the composite films were measured using a texture analyzer  according to Li et al. [TS (MPa) and EAB (%) were calculated as follows:F is the maximum load (N) and A is the initial cross-sectional area of the specimen (mm2). ΔL is the change in initial length up to the breakpoint, and Tensile strength  and puncture deformation (PD) were measured according to Jiang et al. [PS (N/mm) was calculated as follows:F is the maximum puncture force (N) and S is the thickness of the film (mm). Six replicate measurements were performed for each sample.Puncture strength  of the protein spectrum was curve-fitted using Peakfit software, v. 4.12 . For each region analyzed, a linear baseline was established, and the absorbance was normalized with respect to the peak maximum, to avoid undesirable intensity variations. To obtain reasonable fits, Fourier deconvolution was chosen to determine the values of the peak positions with several trials [2 of 0.999.Film samples were measured as described by Stoll, Rech, Flôres, Nachtigall, and de Oliveira Rios  using a l trials . All theFilm samples were measured at room temperature using an X-ray diffractometer  capable of monochromatic Cu-Kα radiation. The equipment was set at 40 kV and 40 mA. The scanning range of the diffraction angle (2 θ) was 4–48° with a step size of 0.02° and a scanning speed of 0.02°/0.1 s.The morphology of the cross-sections of the film samples was investigated via SEM  at an accelerating voltage from 2 to 5 kV.The oral processing assessment was performed by a panel of 14 trained participants  selected on strict dental criteria, who were graduate students majoring in food science. This study was approved by the Medical Ethical Committee of China Agricultural University (Project identification code: 20220204).Participants received three training sessions (1 h/session) with the aid of sample standards to learn to identify and score sample-texture attributes  and to spit the boluses into corresponding containers just before deglutition, as described in a previous study by Lin et al. . They weSix ice creams packed with different packaging films were displayed in an EyeSo Ec-80 eye tracker  to further monitor the visual process of choosing each packed product by participants. The distance between the ET device (the 21″ Full HD screen resolution: 1920 × 1080) and the participants’ eyes was about 60 cm. At the formal measurement, participants were asked to follow the instructions: “Watch the image and use the mouse to select your favorite product.” The participants determined the duration of sample watching. ET metrics included total fixation duration and heatmap distribution . The avep < 0.05) was conducted using GraphPad Prism 7.04 . Spearman’s correlation analysis and principal component analysis (PCA) were used to understand film oral sensory characteristics, physicochemical properties, and the preference of the participants for these films. Spearman’s correlation coefficients were used to determine the relationships at a significance level of p < 0.05 using the SPSS software v. 22 . In PCA, varimax rotation on the correlation matrix using the SPSS software v. 22 and Kaiser criterion  was applied to identify the principal components.Analysis of variance (ANOVA) was used to detect significant differences among films, and Bonferroni’s multiple comparisons test . This was attributed to the moisture migration and the loss of hydrogen bonds between water and protein that caused localized matrix weakening to the protein film network structure, resulting in the decline of the rigidity of the films [Furthermore, FT treatment reduced the TS and PS of WPI-1 and WPI-2 but increased the EAB and PD , regardless of treatment. Indeed, colorants had the ability to decrease or interfere with the film network structure through H-bonding or other chemical bonds [As for the WPI-3 film and its colored versions B,D,F,H, al bonds ,4,30. ThX-Ray diffractograms of WPI-1, WPI-2, and WPI-3 films are compared in −1 and amide II (N-H deformation) at 1650 cm−1, respectively [The FTIR spectra of the WPI-blended films after non-FT and 7-FT treatments are shown in ectively . There wectively . HoweverWith FT treatment , the ranp < 0.05). After FT treatment, compared with non-FT treatment for WPI-1 and WPI-2, the WVP and SR of WPI-1 and WPI-2 were significantly increased (p < 0.05), and the MC of WPI-1 and WPI-2 were significantly decreased (p < 0.05). According to the work of Diefes et al. [p > 0.05), which was mainly attributed to the inhibitory effect of CNC on size and formation of ice recrystallization. However, the SR of WPI-3 changed significantly (p < 0.05), indicating that the IRI effect of CNC was limited and could not fully control the swelling of the film damaged by residual frozen water [The MC, WVP, and SR of the WPI-blended films after non-FT and 7-FT treatment are shown in s et al. , Graivers et al. , and Phas et al.  moistureen water . The proSpearman’s correlation coefficients of all films were compared after non-FT A or 7-FTOn the other hand, with FT treatment B, hardneThe first two principal components of the PCA for the WPI films after non-FT A and 7-FAfter FT treatment, packaged ice creams with different films still had a good sealing and no difference in appearance compared with those of the non-FT treatment C. TexturTo further understand the participants’ acceptance of each packed product, the visual distribution of the participants was analyzed by an ET system. Compared with native WPI packaging, the participants had a longer fixation duration and higher preference levels for colored packaging B. CorresFT treatment reduced the mechanical strength and MC and increased the WVP and SR through physical damage to the film network structure. FTIR measurements showed that, due to the moisture migration, the hydrogen bonds were disrupted, causing the conversion of α-helix and β-sheet to random coil structures. XRD analysis indicated that FT treatment reduced the protein crystallinity of WPI-based films. CNC addition effectively attenuated these effects. Colorant effects on the physicochemical properties of the packaging films were minor due to the low amount used; however, future work should focus on these effects. Notably, although CNC addition improved the physicochemical properties of the films, it reduced their oral tactility preference. According to a combination of sensory data and ET analysis, incorporating colorants remedied this downside and achieved a high overall acceptance of the products. Hence, the key to the development of edible packaging applications in FT environments lies in obtaining a good FT stability and sensory acceptability.


The complex of polysaccharides of the grain transforms during processing and modifies the physical and chemical characteristics of bread. The aim of the research was to characterize the changes of glucans, mannans and fructans in hull-less barley and wholegrain wheat breads fermented with spontaneous hull-less barley sourdough, germinated hull-less barley sourdough and yeast, as well as to analyze the impact of polysaccharides on the physical parameters of bread. By using the barley sourdoughs for wholegrain wheat bread dough fermentation, the specific volume and porosity was reduced; the hardness was not significantly increased, but the content of β-glucans was doubled. Principal component analysis indicates a higher content of β-glucans and a lower content of starch, total glucans, fructans and mannans for hull-less barley breads, but wholegrain wheat breads fermented with sourdoughs have a higher amount of starch, total glucans, fructans and mannans, and a lower content of β-glucans. The composition of polysaccharides was affected by the type of flour and fermentation method used. Hordeum vulgare L.) has been cultivated for centuries all over the world, thanks to its diversity and ability to adapt to the different conditions of climate and soil [Barley  of the Kornelija grain variety was obtained from Stende Research Center . Wholegrain wheat flour was purchased from AS Rigas dzirnavnieks .The hull-less barley  , sugar , salt , barley malt of moisture 9%, diastatic power 250 WK .The research was carried out in the scientific laboratories of the Faculty of Food Technology of the Latvia University of Life Sciences and Technologies.Before the germination or sprouting, grains were rinsed and soaked in water at 22 °C for 24 h. The germination was performed in a climatic chamber  at 35 ± 1 °C, maintaining a constant relative air humidity of 80%. Germination time was up to 24 h. Germinated grains were dried up to 4 h in the universal oven  at 60 ± 1 °C .Hull-less barley (HB) and germinated hull-less barley (HB-G) grains were milled using a Hawos laboratory mill . Obtained flour  was sieved through AS 200 sieves . Resulting flour particle size was 160–710 µm, of which 50% was on the sieve 450 µm, 28% was on the sieve 315 µm and 10% above 710 µm.Spontaneous hull-less barley sourdough (HBS-S) and spontaneous germinated hull-less barley sourdough (HBS-G) were prepared under laboratory-controlled conditions. For the preparation of spontaneous sourdough, the three-stage procedure was used. The preparation of HBS-S was done at optimal fermentation conditions established in previous studies  and descCharacterization of sourdoughs: for HBS-S—lactic acid bacteria 8.0 log CFU (colony forming units)/g; yeast ca 7.0 log CFU/g, pH 3.8, for HBS-G—lactic acid bacteria ca 8.0 log CFU/g; yeasts ca 6.5 log CFU/g, pH 3.9.Hull-less barley breads (HBB) and wholegrain wheat breads (WWB) were prepared in three different ways: fermented with spontaneous hull-less barley sourdough (HBB-S and WWB-S), fermented with spontaneous germinated hull-less barley sourdough (HBB-G and WWB-G) and with yeast fermentation (HBB-Y and WWB-Y).For sourdough bread  preparation flour, spontaneous sourdough , salt, sugar, barley malt and water were used. For bread with yeast fermentation (HBB-Y and WWB-Y) flour, yeast, salt, sugar, barley malt and water were added. Composition of ingredients of HBB and WWB was according to the recipe in The samples of each bread were lyophilized in a freeze-dryer FT33  maintaining −40 °C and 6.4 Pa in a condenser chamber for 72 h. Lyophilized samples were milled using a Foss Knifetec 1095 Mill .2O; 1000—converts mg to g; 100—converts to %.The content of the total glucans and mannans was measured from flours and from lyophilized bread samples using a previously published method  with modStarch analysis was performed in accordance with EN ISO 10520:2001. The content of β-glucans was determined according to the ICC Standard Method No. 168 using Assay kit . Total dietary fiber (TDF) was determined according to the AOAC 985.29.Fructans were determined in lyophilized samples using Megazyme kit K-FRUC  at 410 nm .The volume of breads was determined by rapeseed displacement according to the AACC Method 10-05.01 . SpecifiBread porosity was determined using Zhuravlev device  according to a previously described method by Cizeikiene et al. .Analysis of bread texture was performed by means of a TA.HD.Plus texture analyzer  according to AACC 74-09 with some modifications . Triggerp < 0.05) in chemical and physical parameters of the samples were determined using pairwise Tukey’s honest significant difference (HSD) test.The chemical and physical parameters of HBB and WWB breads were subject to statistical analysis. Statistically significant differences  and correlation analysis were used to determine relationships between chemical and physical parameters and breads samples. Data transformation, analysis and visualization were performed on a custom processing pipeline built using open source Python 3.8 and its libraries NumPy , Pandas The original complex of polysaccharides of the grain changes during processing. This is due to different mechanical processes, hydrolysis and bioprocessing, such as germination and fermentation, therefore it has an effect on the quality of bread in different ways. Significant changes of these substances happen as a result of the activation of the endogenous enzymatic system of the flour, as well as the influence of biochemical transformations caused by microorganisms during fermentation .Starch and non-starch glucan type polysaccharides, mainly β-glucans, are included in the total complex of glucan compounds ,9. In ceIn the result of the metabolic activity of lactic acid bacteria in sourdough and dough, there is a possibility that the microbial glucan type exopolysaccharides can also be produced with different structures and characteristics. The β-glucans of grains and the microbial glucan type exopolysaccharides influence the technological properties and nutritional value of bread, because they potentially act as hydrocolloids and prebiotics ,26,46. TTDF content in different varieties of hull-less barley can be between 11% and 20% . In wholIn different barley species, the content of β-glucans varied between 4 and 9% , but in The content of mannans in WWF flour was 7.29 ± 0.20% dw. In hull-less barley flour (HBF) the content of mannans was 6.29 ± 0.41% dw. In the samples of hull-less barley bread HBB-S and HBB-G, the content of mannans had decreased until 5.23 ± 0.36% and 5.24 ± 0.61% dw, respectively. In the samples of wholegrain wheat bread WWB-S and WWB-G, the content of mannans was not significantly different from flour, 6.92 ± 0.55% and 7.45 ± 0.56% dw, respectively. However, in the sample WWB-Y, the content of mannans was the lowest—5.01 ± 0.24% dw. Between the samples WWB-S and WWB-G, there was no significant difference in the content of mannans. The highest content of mannans was in WWB-S, WWB-G and it was significantly higher than in HBB-S, HBB-G and WWB-Y.The highest content of fructans was found in wholegrain wheat flour (WWF)—1.47 ± 0.22% dw. It did not significantly differ from the content in hull-less barley flour—1.44 0.07% dw. Ispiryan and co-workers (2020) showed similar results of the content of fructans in wheat and barley flour . The amo± 0.00%, and this was significantly lower than in hull-less barley flour (HBF). Due to the increased enzymatic activity, the amount of fructans could either be synthesized or degraded ,51,52. TSaccharomyces cerevisiae cells during fermentation [Results  showed tentation ,53. A loentation ,15,50. TThe behavior of b-glucans, fructans and mannans is similar in barley breads, indicating a decrease of content during the processing of breads.As a result of flour processing the complex of polysaccharides changes, and it influences the technological properties of bread. The results of crumb hardness, stickiness, volume, specific volume and porosity of breads are presented in 2. The scientists have mentioned that fructans are the source of fermented sugars, and the products of their hydrolysis forms a significant part of CO2 in the first stages of dough fermentation, which could also influence the porosity of bread [The specific volume was the lowest in the hull-less barley bread HBB-S and HBB-G. The volume and the specific volume of WWB-Y was significantly higher than that of wheat and barley breads with sourdough. In this research, the preparation of barley bread HBB-Y was considered unacceptable because of the highly dense texture without the increase in volume, and without the signs of yeast metabolism. It was not possible to determine the porosity of HBB-Y. The specific volume was similar for both WWB-S and WWB-G breads, and HBB-S and HBB-G breads. However, other scientists have indicated the highest porosity and specific volume of wholegrain wheat bread by using wholegrain wheat spontaneous sourdough . The higof bread ,53. The of bread .2 [In breads WWB-S and WWB-G fermented with sourdough, the porosity was higher than that observed in HBB-S and HBB-G. Adding the germinated barley sourdough resulted in the higher porosity and larger volume both in barley and wheat breads, which can be explained by an increase of enzymatic activity during the time of germination. This, in turn, impacts the increase of starch degradation and yeast metabolism, and the release of CO2 ,55.In the spontaneous sourdough hull-less barley breads, the crumb was less porous and more compact a in compThe wholegrain wheat bread with yeast fermentation showed the best technological characteristics—crumb hardness, specific volume and porosity. However, it had the lowest content of soluble non-starch polysaccharides such as β-glucans, mannans and fructans, but with the highest total dietary fiber content. The use of spontaneous barley sourdough for wholegrain wheat bread dough fermentation reduced the specific volume and porosity, but did not change the hardness significantly. However, the content of β-glucans was significantly increased. For barley breads HBB-S and HBB-G, the technological characteristics were similar, and there were no significant differences in the content of non-starch polysaccharides.Principal component analysis (PCA) was performed to determine the relationship between the chemical parameters and physical parameters of WWB a,b and HPCA differentiated between HBB and WWB according to chemical a and phyCorrelation analysis between chemical and physical characteristics of HBB and WWB breads is shown in Spontaneous hull-less barley sourdough and spontaneous germinated hull-less barley sourdough can be used to prepare barley and wholegrain wheat bread. From the viewpoint of bioactive polysaccharides, the composition of bread fermented with spontaneous hull-less barley sourdough was similar to the composition of bread fermented with spontaneous germinated barley sourdough. The method of fermentation influenced the content of fructans and β-glucans in bread. In the wholegrain wheat bread with yeast fermentation, the content of fructans decreased faster in comparison to fermentation with barley sourdough. When using the barley sourdough, the content of β-glucans in the wholegrain wheat bread increased twice, but nevertheless it was lower than in the barley sourdough bread. The wholegrain wheat sourdough bread was characterized by a higher content of fructans and mannans, and the same amount of total dietary fiber as in barley sourdough bread.The use of barley sourdough improved the volume, porosity, and reduced hardness of barley bread. Bread made with germinated barley sourdough had a higher hardness and porosity compared to bread with spontaneous barley sourdough. Spontaneous hull-less barley sourdough and germinated hull-less barley sourdough had a negative effect on the physical properties of breads. In comparison to wholegrain wheat bread prepared with yeast, wholegrain wheat breads fermented with sourdough had a higher content of polysaccharides.


The detection of miRNA shows great promise in disease diagnosis. In this work, a ratiometric fluorescent biosensor based on multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes was selected as the target. MWCNTs@Au NCs performs the dual functions of fluorescence quencher and internal fluorescence reference. In the absence of miR-92a-3p, an Atto-425-modified single-stranded DNA probe is adsorbed on MWCNTs@Au NCs, resulting in the quenching of Atto-425. In the presence of miR-92a-3p, the duplex is formed by hybridization of the probe and miR-92a-3p and leaves the MWCNTs@Au NCs, resulting in the fluorescence recovery of Atto-425. DSN can cleave the probe and result in the release of miR-92a-3p. The released miR-92a-3p can hybridize with other probes to form a signal amplification cycle. The fluorescence of MWCNTs@Au NCs remains stable and constitutes a ratiometric fluorescence system with that of Atto-425. A detection concentration interval of 0.1–10 pM and a limit of detection of 31 fM was obtained under optimized measurement conditions. In addition, the accuracy of the biosensor was validated by detecting the concentration of miR-92a-3p extracted from clinical exosome samples. MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs of 18–24 nucleotides that play key roles in many biological processes, including cell proliferation, differentiation, stress resistance and apoptosis by regulating gene expression at the post-transcriptional level . NumerouEffective detection of miRNAs is challenging due to their small size, low expression level and high sequence similarity ,11. Rece3N4, MoS2, gold nanoparticles (Au NPs) and multi-walled carbon nanotubes (MWCNTs) have been widely used in biosensing [Fluorescence-quenching nanomaterials such as Mxenes, graphene oxide, g-Cosensing ,26,27,28osensing ,30,31. Fosensing ,33. To oosensing ,35,36.In this work, we successfully synthesized multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) with dual functions of fluorescence emission and quenching by linking Au NCs to MWCNTs through Au–S bonds. A sensitive and selective ratiometric fluorescent biosensor based on MWCNTs@Au NCs and DSN-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes served as the target in view of its high abundance in exosomes . The morAll serum samples were obtained from healthy controls and CRC patients at the Second Hospital of Shandong University. This work was approved by the ethics committee of the Second Hospital of Shandong University.4·4H2O, AR grade), glutathione reduced , β-mercaptoethylamine , concentrated sulfuric acid  and concentrated nitric acid  were purchased from Sinopharm Chemical Reagent Co., Ltd. Multi-wall carbon nanotubes , N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride , N-hydroxysuccinimide , Tris-HCl buffer  and MES buffer  were purchased from Shanghai Macklin Biochemical Co., Ltd. . DSN was purchased from Evrogen JSC. The probe was purchased from Sangon Biotech (Shanghai) Co., Ltd. . The miR-92a-3p and mismatched sequences were purchased from Shanghai Genepharma Co., Ltd. . Ultrapure water was used in all experiments. All oligonucleotide sequences are listed in Tetrachloroauric(III) acid tetrahydrate  acid tetrahydrate aqueous solution (4 mM) was added to a three-necked flask and stirred at 25 °C for 5 min under a nitrogen atmosphere. Then, 10 mL glutathione reduced aqueous solution (6 mM) was injected and stirred for 10 min. Subsequently, the solution was refluxed at 100 °C for 12 h. The purified Au NCs aqueous solution was obtained by dialysis with ultrapure water for 24 h.For carboxylation of MWCNTs , MWCNTs For conjugation of Au NCs and MWCNTs, MWCNTs (10 mg), MES buffer , EDC (10 mg) and NHS (10 mg) were dispersed in 10 mL ultrapure water and stirred at 30 °C for 0.5 h to activate the carboxyl of the MWCNTs. Next, Tris-HCl buffer  and β-mercaptoethylamine (10 mg) were added and stirred for 12 h. Subsequently, the solution was dialyzed with ultrapure water for 24 h to obtain thiol-modified MWCNTs. Finally, 10 mL Au NCs aqueous solution (0.39 mg/mL) was added to the above solution and stirred for 24 h to obtain MWCNTs@Au NCs. The concentration of MWCNTs@Au NCs was calculated to be 0.7 mg/mL by weighing the mass of the MWCNTs@Au NCs collected by freeze-drying.Probe  and Tris-HCl buffer  were added to MWCNTs@Au NCs solution  and maintained at 25 °C for 40 min. Then, the target (5 μL) with different concentrations, DSN master buffer  and DSN (0.2 μL) were added and incubated at 45 °C for 2 h. After that, DSN stop solution  was added and kept for 10 min to inactivate DSN. The solution volume was quantified to 2 mL by adding ultrapure water. The fluorescence intensities were collected using a fluorescence spectrophotometer. The excitation wavelength was 430 nm, and the slits for both excitation and emission were set to be 15 nm. The fluorescence spectra in the range of 465–700 nm were collected. All measurements were performed in parallel three times, and the averages were reported.ExoQuickTM Exosome Precipitation Solution  was used to extract exosomes from 0.3 mL serum according to the manufacturer’s protocols. The marker proteins CD63 and CD9 of exosomes were analyzed by Western blotting. Total RNAs of exosomes were extracted by miRNeasy Micro Kit (50)  according to the manufacturer’s instruction. Total RNA solution was quantified to 30 μL by adding ultrapure water.The morphology of MWCNTs, MWCNTs@Au NCs and exosomes were observed by a transmission electron microscope . The zeta potentials of MWCNTs and MWCNTs@Au NCs were measured by a zeta potential analyzer . The Fourier transform infrared spectra (FTIR) of MWCNTs and MWCNTs@Au NCs were collected by a Fourier transform infrared spectrometer . The fluorescence properties of MWCNTs@Au NCs were measured by a fluorescence spectrophotometer . The ultraviolet–visible (UV–vis) absorption spectra of MWCNTs and MWCNTs@Au NCs were collected by an ultraviolet–visible spectrophotometer .® Green Premix Pro Taq HS qPCR Kit AG11701  on a CFX96TM Real-Time System . The calibration line was drawn based on the real-time fluorescence curves obtained at different concentrations of miR-92a-3p.RT-qPCR was used as the reference to test the accuracy of the ratiometric fluorescent biosensor. A total of 3.75 μL of RNAs were reverse transcribed by Mir-X™ miRNA First-Strand Synthesis  on T100TM Thermal Cycler  to synthesize cDNAs. RT-qPCR was carried out by SYBRMWCNTs@Au NCs were synthesized following the procedure illustrated in −1 attests to carboxyl existing in the spectrum of MWCNTs, implying that the MWCNTs are rich in carboxyl. In the spectrum of MWCNTs@Au NCs, a characteristic band at 3765 cm−1 attributed to amide appears, while that of carboxyl disappears, indicating that the β-mercaptoethylamine has been modified onto MWCNTs. In addition, a characteristic band at 1645 cm−1 appeared in the spectrum of MWCNTs@Au NCs, demonstrating the successful conjugation of MWCNTs and Au NCs [Conjugation between MWCNTs and Au NCs was further confirmed by FT-IR analysis. As can be seen from d Au NCs . The zetd Au NCs .As depicted in The fluorescence signals of different material systems were measured to evaluate the feasibility of the biosensing b. The fl0)482/F625 value is obtained at 45 °C, while a lower or higher temperature results in a decreased (F-F0)482/F625 value due to inefficient hybridization between probe and miR-92a-3p or reduction in the activity of DSN [The effect of the amount of MWCNTs@Au NCs on the fluorescence signals of the biosensing system was investigated. As shown in y of DSN . Thus, 40)482/F625 values corresponding to mismatched sequences are significantly lower than that of miR-92a-3p, indicating that mismatched sequences cannot give rise to efficient fluorescence recovery of Atto-425. According to the regression equation, the measured concentrations of SMT, TMT and NCT are 0.16, 0.15 and 0.13 pM, respectively, which are much lower than their actual concentrations. This indicates the proposed biosensor can selectively detect miR-92a-3p.The sensitivity of the proposed biosensor to miR-92a-3p was measured under optimized conditions. Before performing the exosomal miR-92a-3p assay, exosomes were extracted from human serum, and their internal miR-92a-3p was obtained by cleaving the exosomes. At first, the morphology of exosomes was characterized by the TEM. They are spherical and exhibit dimensions in the range of 30–110 nm a. AccordTo verify the accuracy of our fabricated biosensor, RT-qRCR and the ratiometric fluorescent biosensor were used in parallel to detect the concentration of miR-92a-3p extracted from exosomes. As shown in In conclusion, a ratiometric fluorescent biosensor based on MWCNTs@Au NCs and DSN-assisted signal amplification was fabricated for the detection of CRC-associated miR-92a-3p extracted from exosomes. Because of the fluorescence reference capability of MWCNTs@Au NCs, this biosensor is stable enough to detect clinical samples. With the introduction of DSN-assisted signal amplification, this biosensor exhibits high sensitivity and a limit of detection as low as 31 fM. Furthermore, the specific cleavage characteristic of DSN endows this biosensor with high selectivity, even against single-base mismatched targets. Furthermore, because of the flexibility of sequence modification, this biosensor is able to detect other RNAs by simply changing the sequence of the probe. In view of these advantages, our fabricated ratiometric fluorescent biosensor shows the possibility of achieving early diagnosis for diseases by detecting miRNAs.


For those with PD-L1 level < 50%, survival was better in the pandemic cohort than the pre-pandemic cohort: hazard ratio (HR) 0.64 . However, for those with PD-L1 level ≥ 50%, survival was not better in the pandemic cohort: HR 1.17 . We found no statistically significant impact of the pandemic on survival among patients treated with pembrolizumab plus chemotherapy. Conclusions: The COVID-19 pandemic was associated with an increase in survival among patients with lower PD-L1 expression who were treated with pembrolizumab monotherapy. This finding suggests an increased efficacy of immunotherapy due to viral exposure in this population.Background: Pembrolizumab monotherapy or pembrolizumab plus chemotherapy has become an important frontline treatment for advanced non-small cell lung cancer (NSCLC). To date, it remains unclear how the coronavirus disease 2019 (COVID-19) pandemic impacted the treatment outcome. Methods: A quasi-experimental study was conducted based on a real-world database, comparing pandemic with pre-pandemic patient cohorts. The pandemic cohort consisted of patients who initiated treatment from March to July 2020, with follow-up through March 2021. The pre-pandemic cohort consisted of those initiating treatment between March and July 2019.The outcome was overall real-world survival. Multivariable Cox-proportional hazard models were constructed. Results: Analyses included data from 2090 patients: 998 in the pandemic cohort and 1092 in the pre-pandemic cohort. Baseline characteristics were comparable, with 33% of patients having PD-L1 expression level ≥50% and 29% of patients receiving pembrolizumab monotherapy. Among those treated with pembrolizumab monotherapy (N = 613), there was a differential impact of the pandemic on survival by PD-L1 expression levels ( Pembrolizumab, a PD-1 checkpoint inhibitor, has become a common frontline treatment for patients with advanced non-small cell lung cancer (NSCLC). Pembrolizumab monotherapy produces a survival outcome that is better than chemotherapy for patients with tumors expressing PD-L1 level ≥ 50% . In addiThe COVID-19 pandemic may have affected cancer patients in several ways. First, during the pandemic, medical resources may have been diverted to COVID-19-related needs, resulting in a difficult access to cancer care and delayed treatment . Second,Understanding the differential effect of SARS-CoV-2 infection on cancer treatment may help the oncology community better prepare for future pandemics. Since advanced lung cancer is a prevalent disease and pembrolizumab is a commonly used treatment for this indication, the information may have a significant public health implication. Because a randomized study will not be feasible for this purpose, we utilized a large, population-based database to conduct a quasi-experimental study, with the pandemic serving as a natural experiment. We characterized the impact of the COVID-19 pandemic on the survival of advanced NSCLC patients during pembrolizumab-based regimens by comparing patient cohorts in the year before and the year of the pandemic. We also investigated diverse patient subgroups for differential impact of the pandemic.The study received approval from the institutional scientific review committee and was exempted from the institutional review board on the basis of the database being deidentified. This study utilized the nationwide, deidentified electronic health record-derived, Flatiron Health database, originating from approximately 280 oncology clinics representing approximately 800 sites of care across the United States. The Flatiron Health database is a longitudinal database, comprising patient-level structured and unstructured data, created via technology-enabled abstraction. This study sought to compare the cohorts of advanced NSCLC patients treated in 2019 (pre-pandemic cohort) and in 2020 (pandemic cohort). Patients in the pandemic cohort initiated treatment between 1 March and 31 July 2020 and was followed through 1 March 2021. To minimize any seasonal trend, the comparator cohort was selected from the exact period in the year immediately before the pandemic. The pre-pandemic cohort consisted of those who initiated treatment between 1 March and 31 July 2019 and was followed through 1 March 2020. To minimize potential effects of the pandemic on the outcome of the pre-pandemic cohort, follow-up was censored on 1 March 2020, which marked the beginning of the community COVID-19 outbreak in the United States.ALK or EGFR were excluded from analyses since frontline pembrolizumab was not indicated for this population. Furthermore, those with delayed initiation of treatment beyond 120 days from the date of advanced NSCLC diagnosis were excluded according to the recommended database rule. Real-world overall survival is defined as an interval between frontline treatment initiation and last follow-up or death. Since the date of death in this database was available only in a month-year format to protect patient confidentiality, the first date of the month after the death month was utilized as the date of death for calculation purposes. Frontline therapy was defined as the first systemic anticancer treatment received since the diagnosis of advanced NSCLC. The performance status was from the Eastern Cooperative Oncology Group (ECOG). Biomarker information was abstracted from unstructured reports scanned into electronic medical records at each medical practice. Geographic location was classified by the state of residence according to the United States Census Bureau.Cohorts were selected from advanced NSCLC patients who started pembrolizumab, with or without chemotherapy, during the specified periods. Patients with alterations in targetable p < 0.05, and all p-values were two-tailed. Statistical analyses and graphics were carried out using SPSS version 26  and SAS version 9.4 .Descriptive statistics including median and range were used for continuous variables, and count and proportion were used for categorical variables. The nonparametric Mann Whitney test was utilized to compare continuous variables, while the Pearson chi-square test was used to compare categorical variables. Survival was estimated using the method of Kaplan–Meier. The log-rank test was used to compare the survival between pre-pandemic and pandemic cohorts. Cox proportional-hazards regression models were fitted to derive hazard ratios (HR) as well as the corresponding 95% confidence interval (CI) and to investigate the interaction between variables. The significance level was set at Based on study criteria , data frp = 0.31.At the time of analysis, 935 deaths had occurred. Including both cohorts, the median real-world overall survival was 10.7 months (95% CI: 10.2–11.2 months). There was no statistically significant difference in the survival between the pre-pandemic and pandemic cohorts. In the pre-pandemic cohort, the median survival was 10.4 months (95% CI: 9.8–11.1), compared with 11.1 months (95% CI: 10.4–11.7) in the pandemic cohort, There were 613 patients treated with pembrolizumab monotherapy: 307 patients in the pre-pandemic cohort and 306 patients in the pandemic cohort. Among them, PD-L1 expression level was available from 542 patients. Most patients who received pembrolizumab monotherapy had a high PD-L1 expression level. The proportion of patients with PD-L1 ≥ 50% was similar across cohorts: 168 out of 267 patients (62.9%) in the pre-pandemic cohort and 173 out of 275 patients (62.9%) in the pandemic cohort.p = 0.31. However, when considering subgroups of patients based on their PD-L1 expression level, a significant interaction between the cohort and PD-L1 expression level was found (p-interaction = 0.02). Among those with PD-L1 < 50%, the median survival was 10.1 months (95% CI: 8.5–11.7). The survival was significantly better in the pandemic cohort than the pre-pandemic cohort: hazard ratio (HR) 0.64   in the pre-pandemic cohort, compared with 11.1 months (95% CI: 10.5–11.7) in the pandemic cohorts,  0.0283) A. On the 0.0283) B. Additi 0.0283) .p = 0.01. However, there was no significant difference in the proportion of those with body mass index ≥ 30: 24.4% vs. 20.9%, p = 0.29, respectively.Among the group of patients treated with pembrolizumab monotherapy, a multivariable analysis was performed to identify independent predictors of survival by integrating the variables: PD-L1 expression level, age, diagnosis to treatment time, sex, body mass index, race, ECOG, state location, smoking history, payers, time period, interaction between time period, and PD-L1 expression level, as well as histology. There were 474 patients with a complete dataset remaining in this subgroup analysis. Using a backward stepwise variable selection process, the variables ECOG and body mass index were identified as independent predictors of survival. Patients with ECOG zero or those with body mass index ≥ 30 had a better survival rate when compared with others: HR 0.56 (95% CI: 0.37–0.90) and HR 0.65 (95% CI: 0.46–0.92), respectively. We also investigated the proportion of patients with ECOG zero and body mass index ≥ 30 across the pre-pandemic and pandemic cohorts. There was a higher proportion of patients with ECOG zero in the pre-pandemic than the pandemic cohort: 86.3% vs. 78.8%, There were 1477 patients treated with pembrolizumab plus chemotherapy: 785 patients in the pre-pandemic cohort and 692 patients in the pandemic cohort. Of these, PD-L1 expression level was available from 1477 patients. Most of the patients had a low PD-L1 expression level. No significant difference was found in the proportion of patients with PD-L1 ≥ 50% between cohorts: 114 out of 600 patients (19.0%) in the pre-pandemic cohort and 105 out of 529 patients (19.2%) in the pandemic cohort.p = 0.47. Moreover, we observed no significant interaction between the cohort and PD-L1 expression level (p-interaction = 0.70), and the survival in the pandemic cohort was comparable to the pre-pandemic cohort across PD-L1 expression levels  and 10.9 months (95% CI: 10.1–11.8) in the pre-pandemic and the pandemic cohorts, respectively, n levels A,B.In this analysis of data derived from diverse oncology practices across the United States, we found that the outcome of patients who underwent frontline pembrolizumab-based treatment was impacted by the pandemic in some subgroup populations. Among those treated with pembrolizumab monotherapy, survival was better in the pandemic cohort than in the pre-pandemic cohort, when the PD-L1 expression level was <50%. However, when the PD-L1 level was ≥50%, the survival was not better in the pandemic cohort. This effect modification by PD-L1 expression level was not observed among those treated with pembrolizumab plus chemotherapy.In the present study, the absence of survival deterioration in the pandemic cohort as compared with the pre-pandemic cohort seems to be at odds with previous studies that have described an increased mortality among lung cancer patients during the COVID-19 pandemic ,16. In oIt is plausible that there is a beneficial effect of SARS-CoV-2 infection in this specific population. Previous literature has suggested a possible synergistic effect of vaccination or viral infection with checkpoint inhibitor immunotherapy. For example, influenza vaccination has been associated with better survival among cancer patients undergoing immunotherapy . It is hTo our knowledge, this study is the first to provide outcome data specific to pembrolizumab-based treatment for lung cancer before and during the COVID-19 pandemic. The advantage of this population-based study includes its relatively large sample size, therefore allowing adequate statistical power to detect the differential effect of the pandemic among population subgroups. However, the study is limited by its non-randomized design, and the data were obtained during routine clinical care, not a controlled research environment, and thus subjected to delayed or missing data. There was a slightly smaller number of patients in the pandemic cohort, suggesting a delay or difficulty in accessing medical care during the pandemic. The dataset is also limited by missing values, and in the subgroup analysis of patients with a complete dataset, the effect modification of PD-L1 expression level by time period was no longer significant Furthermore, important covariates such as confirmation of COVID-19 infection, comorbidity, or socioeconomic status were unavailable. Nonetheless, these confounders should not result in a differential impact of treatment based on PD-L1 expression status. Finally, we do not have data on treatment-related toxicity or cause of death, which could help explain the findings. Nonetheless, the large number of patients helps mitigate statistical power loss from missing data, and the baseline observable characteristics between the pandemic and pre-pandemic cohorts are well balanced, thus reducing the threat of confounders.In summary, this population-based study demonstrated an improvement in real-world overall survival during the COVID-19 pandemic among a specific group of lung cancer patients undergoing pembrolizumab-based treatment: those with PD-L1 expression level < 50% treated with pembrolizumab monotherapy. Future studies will be needed to understand the underlying mechanisms contributing to the survival improvement.


A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.  Forty years ago, Folkman claimed that angiogenesis is essential for tumors to grow beyond essential size  and it iCorynebacterium diphtheria as a single-chain enzyme of 535 amino acids with a molecular weight of 63 kDa [1], [2]. This toxin has two domains namely A and B. B domain is responsible for cellular binding and translocation, while A subunit is responsible for inhibition of protein synthesis by transferring the ADP-ribosyl moiety of NAD+ to the eukaryotic polypeptide elongation factor 2 (EF2)  , 17. DT  2 (EF2) . This to 2 (EF2) , E7777 ( 2 (EF2) , and DT3 2 (EF2) , 22. Amo 2 (EF2) . in vivo assessed in the mouse tumor model. In this study, the diphtheria toxin subunit was genetically fused to mouse VEGF and formed an immunotoxin. This protein was expressed in the bacterial expression system and its biological activity was Gene construction and cloningNdeI and XhoI restriction sites of the vector following ligation by T4 DNA ligase and were transformed into E. coli TOP10F’. The resulting plasmid was named pET-mVEGF-DT. The cloning procedure was confirmed by colony-PCR with T7 universal primers and restriction enzyme digestion. The DT fragment was also cloned and constructed (entitled as pET-DT). Two DNA coding sequences consisting of mouse VEGF (NM_001025257.3) and truncated diphtheria toxin, DT386, genetically fused with a human IgA1 hinge as a linker and synthesized . DNA fragments were sub-cloned into the pET22b expression vector in Immunotoxin expression and purification600 of 0.6 was reached, expression of recombinant protein was induced with 1mM of Isopropyl-beta-d-thiogalactopyranoside (IPTG) and incubated at 37 °C overnight. Recombinant expression was confirmed by SDS-PAGE and Western blotting. Briefly, proteins were separated in 15% SDS-PAGE and were transferred to nitrocellulose paper with the semi-dry method . Blot was blocked by 3% skimmed milk overnight at 4 °C and then incubated with 1:2000 diluted rabbit anti-His antibody and anti-rabbit-HRP conjugated antibody (1:1000). Finally, the blot was developed with 4-chloro-1-naphthol.BL-21 (DE3) bacteria were transformed with pET-mVEGF-DT and pET-DT plasmids. The single colony that was positive in colony PCR and digestion, was inoculated in LB media in presence of selective antibiotics. When OD2PO4, 10 mM Tris-Cl, 8 M Urea, pH 8). Sonication was used to lyse the cells. The soluble fraction was separated by centrifugation and 0.45-micrometer filter, and the lysate was applied to the Ni-NTA column  which was pre-equilibrated with binding buffer. After washing the column with wash buffer , His tagged portions were eluted by applying elution buffer . The purity of the preparation was verified by SDS-PAGE.Protein purification was performed under denaturation conditions. The cell pellet was collected by centrifugation  and re-suspended in the binding buffer . TC-1 was derived from primary lung epithelial cells of C57BL/6 mice. The right flank of the C57BL/6 mice was shaved, and cells were injected. This mouse was used as stock. Following the establishment of the tumor, the stock mouse was sacrificed and the tumor was dissected into 3 mm3 segments that were then transplanted into 18 mice. Tumor size was monitored three times a week using a caliper according to this equation  mice were purchased from the animal facility of Pasteur Institute of Iran. All animals used in this study were cared for according to the animal care and use protocol of the Pasteur Institute of Iran. The tumor was induced in these mice by injection of 10equation : V=L×W2×Treatment study3 and continued for 2 months. The mortality was monitored during this period. For each injection, the control group received 100 µl PBS; the DT group received 100 µg of DT toxin, and the test group received 100 µg of mVEGF-DT. The final volume for each injection was 100 µl. The TC-1 tumor-bearing C57BL/6 mice were divided into three groups, each group contained 6 mice. Mice were injected intra-tumoral in weekly intervals. Treatment started when tumor volume reached 4 mmEthical standardsAll animal studies have been approved by the Institutional Ethics Committee (Pasteur Institute of Iran). All researchers got acquainted with the ethical methods of working with laboratory animals before starting their research.Statistical analysisP<0.05). GraphPad PRISM software was used for statistical analysis. The survival rate was evaluated by Kaplan-Meier analysis. Two-way ANOVA was used for comparison. The mean of the size of the tumor was calculated within each group, and one-way ANOVA was used to determine the significant difference between groups . There was a significant difference between the mVEGF-DT group and the DT group  and also the mVEGF-DT group and the PBS group  from the fourth week onwards. Also, there was no significant difference between the two groups of DT and PBS by the end of the eighth week . All mice in the mVEGF-DT treated group survived until the end of the experiment . DT protein had increased the survival of the tumor-bearing mice when compared with the PBS group .For periment . There wTM) administered as an antineoplastic agent, interleukin -3 conjugated to the diphtheria toxin (ElzonrisTM) used for the treatment of blastic plasmacytoid dendritic cell neoplasms (Immunotoxins include a target recognition moiety that is connected to bacterial or plant proteinaceous toxins. The target recognition portion can include complete monoclonal antibodies, antibody fragments, or ligands that bind to the receptor on the cell surface. Diphtheria toxin is one of the most widely used toxins for the development of immunotoxins , 25. It eoplasms  and antieoplasms . Immunoteoplasms , prostateoplasms , pancreaeoplasms , and auteoplasms . The speet al., and it was shown that destruction of the vasculature around melanoma and human prostate carcinoma xenografts in mice leads to decreased tumor volume (et al. (Angiogenesis is a critical multi-step process that results in the growth and sprouting of solid tumors. It has been proven that inhibition of angiogenesis, is an effective way to treat solid tumors and is a key factor in the growth of solid tumors. This process is involved in many different physiological processes such as embryonic development and wound healing . Dysregur volume . They sp (et al.  used a D (et al. . DT386 t (et al. . The samThe successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates. Statistical analysis showed that a significant difference was observed between groups. However, it is suggested that the ability of the immunotoxin to detect VEGF be evaluated in flow cytometric and immunohistochemical tests. Furthermore, the level of immune response to immunotoxins in the animal model should be examined.MH, MB, BR, and RM Data analysis and draft manuscript preparation; NS and FK Critical revision of the paper; MB and MH Supervision of the research; HG and AO Data processing, collection, performing experiments; MB Study concept and design.The authors declare that they have no conflicts of interest.


The introduction of direct-acting antiviral drugs (DAAs) has caused a paradigm shift in the management of HCV infection; recent guidelines recommend pan-genotypic drugs  as the first-choice therapy for HCV, and these promise to be effective and safe even in the context of chronic kidney disease. Aim: The purpose of this narrative review is to show the most important data on pan-genotypic DAAs in advanced CKD (CKD stage 4/5). Methods: We recruited studies by electronic databases and grey literature. Numerous key-words  were adopted. Results: The most important pan-genotypic combinations for HCV in advanced CKD are glecaprevir/pibrentasvir (GLE/PIB) and sofosbuvir/velpatasvir (SOF/VEL). Two clinical trials (EXPEDITION-4 and EXPEDITION-5) and some ‘real-world’ studies (n = 6) reported that GLE/PIB combinations in CKD stage 4/5 gave SVR12 rates ranging between 86 and 99%. We retrieved clinical trials (n = 1) and ‘real life’ studies (n = 6) showing the performance of SOF/VEL; according to our pooled analysis, the summary estimate of SVR rate was 100% in studies adopting SOF/VEL antiviral combinations. The drop-out rate (due to AEs) in patients on SOF/VEL ranged between 0 and 4.8%. Conclusions: Pan-genotypic combinations, such as GLE/PIB and SOF/VEL, appear effective and safe for HCV in advanced CKD, even if a limited number of studies with small sample sizes currently exist on this issue. Studies are under way to assess whether successful antiviral therapy with DAAs will translate into better survival in patients with advanced CKD. Background: Hepatitis C virus (HCV) is still common in patients with chronic kidney disease. It has been recently discovered that chronic HCV is a risk factor for increased incidence of CKD in the adult general population. According to a systematic review with a meta-analysis of clinical studies, pooling results of longitudinal studies ( HCV was identified in 1989; since then, HCV has emerged as a major public health problem . In 2020n = 15 studies, n = 2,299,134 unique patients) demonstrated an association between positive anti-HCV serologic status and increased incidence of CKD; the summary estimate for adjusted HR across the surveys was 1.54  (p < 0.0001). However, between-study heterogeneity was observed  [The relationship between chronic kidney disease and HCV infection is bi-directional; HCV infection is both a cause and consequence of chronic kidney disease . AccordiThe introduction of direct-acting antiviral agents (DAAs) has created a paradigm shift in the management of infection by HCV ,4. More Studies were identified by searching electronic databases and sources of grey literature. The literature search was applied to PubMed MEDLINE, EMBASE, and Google Scholar. The following key words were adopted: (‘Hepatitis C Virus’ OR ‘HCV’ OR ‘Hepatitis C’) AND  AND . We performed pooled quantitative summary estimates of the sustained viral response (SVR) and discontinuation rates of antiviral therapies (pan-genotypic DAAs for HCV in advanced CKD) across individual studies using the inverse-variance method. A random-effects meta-analysis was conducted ,6. Outcoper se on death rate, irrespective of the role of covariates. Pooled RRs and their 95% CIs were estimated by the weighted inverse of their variance. Heterogeneity was evaluated using Der Simonian and Laird’s Q test and quantified by calculating the proportion of the total variance attributable to between-study variance (Ri). We computed both fixed- and random-effects models but used the latter in case of large heterogeneity. We adopted HEpiMA, a novel software program that carries out a complete study of heterogeneity of study effects [i and CVB were used. Heterogeneity was considered substantial if Ri was ≥ 0.75. A two-sided p-value of <0.05 was considered statistically significant. Observational studies that compared the all-cause mortality in anti-HCV positive patients who received antiviral therapy compared with those who did not were also retrieved. The aRR was obtained in each study by multivariate analysis to find the impact of antiviral therapy  effects . Novel aAbundant information exists on the epidemiology of hepatitis C virus infection in patients with end-stage renal disease. In 2012–2015, anti-HCV antibody prevalence among prevalent HD patients in the DOPPS was 9.9% overall ; it ranged from 4.1% in Belgium to 20.1% across the Gulf Cooperation Council Countries , whereas >8% of patients receiving HD in China, Italy, Japan, Russia, and Spain were anti-HCV positive . Another survey was conducted among Medicare beneficiaries with HCV undergoing haemodialysis in the United States (2005–2016). A total of 291,663 patients on haemodialysis were enrolled. The prevalence of HCV in patients on haemodialysis was greater than in individuals not on HD (4.2 vs. <1%) . n = 106, 463 unique patients) and found a regional pooled mean estimate of 29.2%  for HCV antibody prevalence among patients on long-term haemodialysis in the Middle East and North Africa (MENA) [Evidence on the prevalence and incidence rates of HCV among patients on long-term dialysis in the emerging world was not satisfactory—several studies with small sample sizes have been published, and recorded prevalence rates of up to around 80% ,10,11,12p = 0.008) and 22.8 ± 1.7 vs. 16.1 ± 1.7 (p = 0.0001). According to logistic regression analysis, detectable HCV RNA in serum was a strong predictor of raised AST (p = 0.0001) and ALT (p = 0.000001) values [It is difficult to make a detailed evaluation of the natural history of HCV infection in patients with chronic kidney disease, particularly those on maintenance dialysis. Various reasons explain this—the natural history of HCV usually spans decades in patients with intact kidneys, whereas dialysis patients have limited life expectancies. In fact, patients with chronic kidney disease have higher morbidity and mortality than the general population, due to aging and comorbidities. HCV infection is frequently asymptomatic, with an apparently indolent course even in patients with advanced chronic kidney disease. Aminotransferase values are lower in patients on maintenance dialysis; thus, it is not easy to recognise the occurrence of liver disease on the grounds of biochemical abnormalities. A total of 506 patients undergoing regular dialysis in northern Italy (Lombardy) were tested by anti-HCV ELISA and PCR assays for the detection of anti-HCV antibodies and HCV RNA in serum, respectively. Serum transaminase values were significantly greater in HCV RNA-positive than HCV RNA-negative patients, 19.3 ± 1.6 vs. 15.7 ± 1.6  values . n = 23 studies; n = 574,081 patients on long-term dialysis). We found that positive anti-HCV serologic status was an independent and significant risk factor for death in the dialysis population. The overall estimate for adjusted mortality  with HCV was 1.26  (p < 0.0001) [p < 0.0001). The overall estimate for cardiovascular death risk was 1.18  (p < 0.0001). Using meta-regression, we observed that the relationship between positive anti-HCV serologic status and all-cause death risk was more evident in surveys with a larger size (p < 0.0001), higher proportion of diabetics (p = 0.0005), and HCV-positive patients (p = 0.001) [The current availability of direct-acting antiviral agents, which are considered to be safe and have high efficacy, precludes the implementation of large observational studies and prolonged follow-up to assess the course of chronic HCV infection in end-stage kidney disease. It has been stated that survival in most patients with stage 1 and 2 CKD is not different from that observed in the general population with intact kidneys. Survival in patients with CKD stages 3–5 is lower than that observed in the general population, and some information has been recently accumulated on the link between positive anti-HCV serologic status and the death rate in the dialysis population. Death can be considered a reliable endpoint in the context of observational studies evaluating the course of HCV over time in patients with intact kidneys or end-stage kidney disease, and some clinical studies have carried out such analyses. We recently conducted a systematic review with a meta-analysis of observational studies ( 0.0001) . We perf= 0.001) . p = 0.005). The study group in such a report was large  [The latest report on this issue was published a few months ago by Ko and coworkers. After adjusting for potential confounding factors, the multivariate Cox regression resulted in a significant association between serum HCV RNA-positive status and death rate in a cohort of haemodialysis patients who started chronic HD after 2002  . The purpose of antiviral therapy of HCV was the achievement of SVR12. SVR12 is the elimination of HCV RNA from serum which persists at least 12 weeks after completing antiviral therapy . Antivirn = 287 unique patients) and the summary estimate for SVR and drop-out rate was 0.60  and 0.18 , respectively. The most common source of drop-out was anaemia (11/46 = 23%) [The evidence in the medical literature supporting the antiviral treatment of HCV in patients receiving long-term dialysis is poor. Prior to the advent of DAAs, IFN-based regimens were the standard of care for the antiviral treatment of HCV. Clinicians had been reluctant to adopt IFN-based regimens for HCV in patients on maintenance dialysis, due to limited efficacy and low tolerability of IFN-based regimens in the haemodialysis setting. The effectiveness and tolerability of combined antiviral therapy (pegylated interferon plus ribavirin) in patients on regular dialysis had been addressed in a meta-analysis of clinical studies. We identified eleven clinical studies  (p = 0.0001), acute kidney failure before renal replacement therapy , and antiviral treatment  were independent predictors of improved survival in patients on maintenance haemodialysis with chronic HCV. Age at HD initiation was linked to lower survival . Soderholm and coworkers  gave ant 0.0001) . Accordin = 2231), 134 (6.01%) patients received interferon and 2097 (93.9%) did not; the mean follow-up duration was 3.22 years  and used propensity score-matched and competing risk analyses to evaluate the long-term patient survival (death rate) of anti-HCV therapy, especially interferon-based therapy, in CKD patients. They observed that the treated cohort had a 29%  (p = 0.011) decrease in death compared with the untreated cohort. Chen and coworkers  made a lp < 0.001).The last report on this topic was published by Perez de Josè and coworkers . They rep < 0.001) . Further study is needed to make more definitive conclusions. All the studies reported in < 0.001) . The res< 0.001) ; however2. In addition, the administration of a single full dose of SOF revealed that the plasma AUC of GS-331007 were 5.6-fold and 6.83-fold higher in patients with CKD stages 4 and 5, respectively, than those with intact kidneys [Sofosbuvir was approved in 2013 and is now the backbone of the most commonly used DAA regimens. SOF is an oral nucleoside analogue and potent inhibitor of the NS5B RNA-dependent RNA polymerase. Upon oral administration, SOF is metabolized (at liver level) to 2′-deoxy-2′-alpha-fluoro-beta-C-methyluridine-5′-monophophate, which undergoes conversion to the active triphosphate form (GS-461203). SOF acts as a HCV RNA chain terminator by inhibiting NS5B RNA-dependent RNA polymerase, which is essential for the replication of the HCV RNA viral genome . The dep kidneys . A pharm kidneys . At the n = 1537 unique patients). The overall frequency of SVR12 was 0.99 ; the pooled frequency of SAEs was 0.09 . Some  clinical studies reported eGFR values at the beginning and end of antiviral therapy, and no consistent changes were recorded [A systematic review of the medical literature with a meta-analysis of clinical studies has been recently published with the aim to assess the effectiveness and tolerability of SOF-based regimens in patients with advanced CKD . The primary end-point was the frequency of sustained viral response (as a measure of efficacy); the secondary outcomes were the rates of SAEs and drop-out due to AEs (as measures of tolerability). We identified 30 clinical studies  for 12 weeks in adults with HCV infection and HCV genotype 1, 2, 3, 4, 5, or 6 and compensated liver disease (with or without cirrhosis) . All patn = 3) and raised serum creatinine (n = 2). Other reasons were cerebral infarction (n = 1), fungal peritonitis (n = 1), patient preference (n = 1), and cardiomyopathy (n = 1). Some ‘real-life’ studies have also been published in patients with advanced CKD and showed SVR12 rates similar to those observed in EXPEDITION-4 and EXPEDITION-5 trials  29,30,3,32,33,34n = 54) or peritoneal dialysis (n = 5) [Borgia et al. performed a phase II single-arm study to treat 59 patients with genotype 1–6 HCV infection on long-term haemodialysis ( (n = 5) . These pn = 7) showed early discontinuation of therapy due to AEs, and the causes were weakness (n = 1), anaemia (n = 1), dizziness (n = 1), and gastrointestinal disorders (n = 4). Some real-world studies ,40,41,42No data have been currently published on the antiviral combination of sofosbuvir/velpatasvir/voxilaprevir (SOF/VEL/VOX) in patients with advanced CKD. Patients with advanced CKD have been defined a ‘special population’ or ‘difficult-to-treat population’, where antiviral therapy has historically been unable to generate high efficacy and safety. DAA-based regimens have revolutionized the management of HCV, and recommended DAA regimens can vary in duration, dosing frequency, pill burden, and co-administration of ribavirin. Pan-genotypic drugs allow the simplification of the management of HCV. These drugs reduce the need for pre-treatment testing, and consequently, the time between HCV diagnosis and therapy initiation is shortened. Pan-genotypic agents promise to be safe and effective, even in patients with advanced CKD, and more studies are needed to confirm and expand such pieces of evidence. An important limitation of the present narrative review was the limited number of included studies and the small number of patients overall. Research aimed to assess whether successful antiviral therapy with DAAs will improve survival of patients with advanced CKD is in progress.


The probe NS-ClO for the recognition of HClO  in vivo with a large turn-on signal (about 860 times) and Stokes shift (about 90 nm), fast response (about 1 min) and good selectivity.Fast-responsive two-photon fluorescent probe NS-ClO for imaging endogenous HClO  When the balance of HClO in the body is destroyed, many molecules such as DNA, RNA, fatty acids, cholesterol, and proteins can react with HClO, which is related to different diseases including neurodegenerative disorders and cancers.2–5 Further research between the HClO level and the pathophysiological process is very necessary. Therefore, it is important to develop a practicable method for monitoring HClO in a physiological atmosphere.Hypochlorous acid (HClO) is a weak acid with oxidizing properties in the reactive oxygen species (ROS) family and it is generated from living immunological cells by oxidising hydrogen peroxide  and Stokes shift (about 90 nm) . This tu1H NMR, 13C NMR and HRMS was performed, and the details are provided in the ESI.†The sulfur atom in phenothiazine is very reactive towards HClO. The fluorescence property of phenothiazine was changed by oxidising a sulfur atom to sulfoxide, as depicted in The spectral properties of probe NS-ClO were studied. There was almost no fluorescence intensity of probe NS-ClO at 450 nm in PBS buffer (pH = 7.4) and DMF v/v = 19/1) at an ambient temperature without the addition of HClO . When di9/1 at anφ = 0.03) using a fluorescein (φr = 0.90 in 0.1 N NaOH) solution.27 However, when different amounts of HClO were added, the obvious turn-on fluorescence enhancement (about 860-fold) exhibited a quantum yield of 0.57 at 450 nm with a detection limit of 0.75 μM . With the addition of HClO (5.0 equiv.), the fluorescence intensity gradually increased when the pH was changed from 1.0 to 8.5 and rapidly decreased from 8.5 to 10.0. The main reason is that the oxidizing properties of HClO decline significantly in alkaline conditions. However, we found that the probe NS-ClO could detect HClO in PBS buffer (pH = 7.4) containing DMF (v/v = 19/1) at the physiological pH (7.4) with about 860-fold enhancement. In other words, this probe can be suitable for biological applications . However, when we added HClO to the detection system, the fluorescence enhancement increased significantly within 1 min. This result indicated that the NS-ClO probe can be applied to monitor HClO with good selectivity compared to other different species , reactive nitrogen species (RNSs) and anions. As listed in  species .Encouraged by the above-mentioned excellent results, we inferred that the highly sensitive and selective two-photon probe NS-ClO could be suitable for imaging HClO in the living biosystem. First, the results of MTT assays proved that the HeLa cell survival rate is very high after treatment with different concentrations of NS-ClO. That is to say, the probe NS-ClO exhibited low cytotoxicity to HeLa cells after one day even at high concentrations (30.0 μM) Fig. S5 and coul29 lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) are added to stimulate macrophages to produce endogenous HClO. As depicted in −1) and PMA (2 μg mL−1) together and then treated with probe NS-ClO (10 μM), obvious fluorescence enhancement was obtained  for 30 min at 37 °C. After scanning by TPM, there was no fluorescence signal in the blue channel  and low cytotoxicity. In addition, the two-photon probe NS-ClO could be used for the detection of HClO in living cells, tissues and fresh zebrafish in vivo. Therefore, the probe NS-ClO can be developed into other functional two-photon probes for the recognition of other analytes and can be applied to investigate the biological and pathological functions of HClO in living biosamples.In conclusion, a fast-responsive fluorescent probe with two-photon excitation, a large turn-on signal (about 860 times) and a large Stokes shift (about 90 nm) for the detection of HClO All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shandong University and experiments were approved by the Animal Ethics Committee of Shandong University according to the requirements of the National Act on the use of experimental animals (China).There are no conflicts to declare.RA-009-C9RA02160D-s001


Self-healing coatings formulated by stimuli-responsive container technology are regarded as a prospective strategy for long-term corrosion protection. However, such types of coatings suffer from low coating adaptability and delays in corrosion protection because the occurrence of corrosion is prior to the release of healants from containers. Herein, we took advantage of the easy hydrolysis of MOF-199 for water-induced self-healing properties. Mixed corrosion inhibitors were loaded into MOF-199 and then incorporated into acrylic coating. The water sensitivity of MOF-199 was investigated and EIS tests were used to evaluate the self-healing performance. Due to the collapse of the porous MOF-199 structure, corrosion inhibitors could be released from MOF-199 with the invasion of water into acrylic coating. The corrosion resistance performance of damaged self-healing coating gradually increased. The metal exposed to artificial defects was well protected due to a barrier formed by corrosion inhibitors. Owing to these merits, this self-healing coating is recommended for use in various fields of engineering for corrosion resistance. Therefore, the PPy/Mo@TiO2 coatings can serve as an excellent self-healing coating and provide long-term corrosion protection. However, some limitations are exposed as well. First, the occurrence of significant local pH changes is due to the formation of OH− at cathodic areas and the hydrolysis of metallic ions at anodic areas during corrosion reaction. At this time, the metal substrate is severely corroded. This severe corrosion is ahead of the release of corrosion inhibitors. Additionally, since some containers can release corrosion inhibitors in response to pH, they may not be very suitable for coatings whose intrinsic environments are acidic or alkaline. For example, the crosslink of the epoxy oligomer usually needs an alkaline curing agent, making the resultant coating alkaline. Therefore, there is still room for the advancement of self-healing coating to solve these challenges.Endowing coating with self-healing functions is considered as a promising strategy for long-term metal corrosion protection. Self-healing coating can heal the microcrack or significantly prolong the life cycle of coatings. In recent years, various strategies have been developed to achieve self-healing effects, including embedment of healant-loaded nanocontainers into the coating ,2,3, shaodextrin ,7, graphodextrin ,9,10,11,odextrin ,13,14, hodextrin ,15, titatrin [2) ,17, etc.trin [2) , Ficus rtrin [2) ,20, 8-hytrin [2) , cerium trin [2) , molybdatrin [2)  etc.). TMetal-organic frameworks (MOFs) are a kind of crystalline porous structure formed by self-assembly of metal ions and organic ligands with the features of high specific surface area and regular tunnel. MOF-199, also known as HUSKT-1, is a subclass of MOFs formed by copper ion coordination with 1,3,5-benzenetricarboxylic acid (BTC) ligands. MOF-199 is prone to acidolysis and hydrolysis. An investigation of water-triggered MOF to release corrosion inhibitors was implemented in this work because the invasion of corrosion media is accompanied with salt neutral water in marine environments. MOF-199 as a container was used to load the corrosion inhibitor and then they were embedded into the coating. When corrosive media penetrates the coating and contact with MOF, the MOF will decompose, leading to the release of the corrosion inhibitor ahead of the local pH change caused by severe corrosion.2Cl2) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . BTC, sodium molybdenum oxide (Mo), and ethanol were obtained from Shanghai Macklin Biochemical Co., Ltd. . CuCl2·2H2O and 1H-Benzotriazole (BTA) were offered by Tianjin Baishi reagent Co., Ltd. . Deionized water was obtained from the Millipore purification system. Q235 carbon steel (CS) was obtained from the Xunjin stainless steel processing center . The commercial clear acrylic resin was kindly provided by Liaoning Pengwei Chemical Paint Co., Ltd. . All reagents were used as received.N, N-Dimethylformamide (DMF), and dichloromethane (CH2·2H2O (0.2319 g) and BTC (0.1765 g) were added in a test tube and then ultrasonically dissolved in 9 mL mixed solvent (water:DMF:ethanol = 1:1:1 in volume). The test tube was tightly sealed by a stopper. The solution was heated to 85 °C under vigorous stirring for at least 24 h. The solution turned from green to blue and precipitation gradually was formed with time. The resultant blue precipitation (MOF-199) was collected by filtration at room temperature and rinsed with DMF twice to remove the remaining reactants. Then the MOF-199 was further adequately rinsed with CH2Cl2 to remove the solvent. The product was finally obtained after drying in vacuum at 60 °C for 24 h. The yield of the obtained MOF-199 was ~80%.MOF-199 was prepared via a typical hydrothermal method. CuClBTA (0.1 g) and Mo (0.1 g) were dispersed in 50 mL ethanol. Then MOF-199 (0.1 g) was added into the mixture under vigorous stirring for 24 h. Then the mixture was collected by filtration and rinsed with ethanol to remove the unstable immobilized corrosion inhibitor. The production was obtained after drying at 50 °C for 24 h.BTA-Mo@MOF-199 (0.1 g) and commercial acrylic coating (2 g) were dispersed in ethanol under vigorous stirring. Then the well-dispersed mixture was rapidly coated on carbon steel by an automatic coating applicator . The coating thickness was ~80 μm. To remove the bubble gas inside the coating as possible, the as-prepared sample was placed in a vacuum for 48 h. The composite coating, denoted as BTA-Mo@MOF-199/acrylic coating, was finally obtained after drying at room temperature for 7 days. The whole process is demonstrated in −1. XRD diffraction patterns of all samples were determined by an X-ray diffractometer (Bruker D8 ADVANCE). The thermogravimetric analysis was performed on thermogravimetry (TG209F1 libra). Samples were heated from room temperature to 900 °C at the rate of 10 °C/min in nitrogen. N2 adsorption/desorption at liquid nitrogen temperature was used to determine specific surface areas and pore volumes of the samples as well as the pore sizes of samples by Micromeritics ASAP 2460. All samples were outgassed at 343.15 K for 12 h under a vacuum of 10−4 Pa before measurements. Electrochemical impedance spectroscopy (EIS) was conducted to investigate self-healing performance of samples by an electrochemical station (Gamry 600) with an amplitude of 20 mV at the frequency range from 105 to 10−2 Hz. . Prior to the self-healing test, samples were damaged to form a scratch with a length of ~2 mm by a scalpel. Then, damaged samples with 1 cm2 as working electrode were immersed in 3.5 wt.% NaCl aqueous solution. Pt and calomel electrodes were used as counter and reference electrodes, respectively.The morphology of the MOF-199 and self-healing coating were observed by Field Scanning Electron Microscope  and the energy dispersive spectrum (EDS) was mapped for element analysis. The FTIR spectrum of the MOF-199 was measured by an infrared spectrometer (EQUINOX 6000) at the wavelength of 4000–500 cm2 H2O and BTC in the mixed solvent. XRD and FTIR were used to characterize the resultant MOF-199. The XRD patterns and FTIR spectrum of MOF-199 are indicated in 2 H2O and BTC), which showed the huge difference in diffraction peaks  of pure MOF-199 is due to the decomposition of the MOF-199 at the temperature range of 200~380 °C [The thermal stability of MOF-199 is necessary to investigate in practical applications. 0~380 °C . This alTo investigate the water sensitivity of MOF-199, we immersed MOF-199 into water and made a comparison in MOF structure. 2 adsorption-desorption isotherms according to IUPAC classification  can also indicate the overall resistance in the electrochemical system for a semi-qualitative analysis of the corrosion resistance. Large |Z|0.01Hz means high resistance in the electrochemical system. For the bare carbon steel, a small semicircle and a low |Z|0.01Hz can be observed in Nyquist and Bode plots  (f means low coating permeability. Qf of the damaged pure acrylic coating increased to 47 μF/cm2 compared with that of the intact one (6.28 × 10−4 μF/cm2) in ct showed a continuous downward trend with time due to the gradual diffusion of aqueous NaCl to the metal interface. Similar to pure acrylic coating, MOF-199/acrylic coating without corrosion inhibitors also fails with the increase of Rct over time. On the contrary, it significantly decreases with time, demonstrating that the pure MOF-199 had no contribution to the self-healing effect  b. For th6 Ω cm2) d. Coating effect . This ev0.01Hz was observed with time, indicating that the impedance of the whole electrochemical system was enhanced with time after being damaged  and corrosion reactions (Qdl) and decreases in Rct. By comparison, these parameters variation of BTA-Mo@MOF-199-incorporated coating is similar to pure acrylic and MOF-199-incorporated coatings before 8 h. However, significant decreases in coating permeability (Qf) and corrosion reactions (Qdl), as well as increases in Rct were observed in 12 h. 0.01Hzwith time can be observed in F and QF refer to the resistance and capacitance of film formed by corrosion inhibitors, respectively. Rct can maintain 105 orders of magnitude with time  to form another film adsorbed on the metal surface. These could depress corrosive media to attack the metal so as to prohibit the further development of the metal corrosion after 12 h. damaged a. In theith time b, showinith time d. Fitted in 12 h d. A signion time c. Due toion time . This is damaged a, resultith time c, reflecIn this work, we synthesized MOF-199 via a hydrothermal reaction of BTC and copper salt. The MOF-199 structure with a high pore volume as well as absorbance capacity can serve as a competent container to load the mixed corrosion inhibitors. The MOF-199 exhibited a certain thermal stability and its decomposition temperature reached 200 °C. The MOF-199 structure is prone to collapse in contact with water due to the metal substitution and the decomposed products showing limited absorbance capacity. So, the BTA-Mo@MOF-199 can rapidly release corrosion inhibitors under water stimuli. After the incorporation of BTA-Mo@MOF-199, the corrosion resistance performance of the damaged acrylic coating will gradually increase with time. In summary, the BTA-Mo@MOF-199 can endow the commercial acrylic coating with water detection and self-healing functions. All raw materials used in the facile fabrication of this self-healing acrylic coating are economically accessible. We believe this type of coating is a promising coating in mass production at the plant level and practical corrosion protection in various engineering fields.